The applicants are aware of various methods for determining pregnancy status, the number of foetuses present and the stage of pregnancy within females (animals and humans). The methods that are used may be divided into two broad categories; physical detection of the foetus and/or pregnant uterus and laboratory-based methods for detecting chemical and/or hormonal changes within the female that are associated with pregnancy.
Physical methods include the manual palpation of the foetus and pregnant uterine structures by an experienced operator. In humans this service is typically provided by medical practitioners and in animals by veterinary surgeons. For example, in cattle, the foetus along with changes to the uterine environment associated with pregnancy can be manually detected by per rectum palpation of the uterus and its contents from around 35 days of pregnancy by an experienced operator. Larger animals (such as horses and cattle) are amenable to per rectal manual palpation of the uterine contents and uterine environment and, as such, pregnancy may be detected at an earlier stage than for animals that cannot be submitted to this process (e.g. humans, dogs, cats, sheep, pigs).
Manual palpation in larger animals by experienced operators allows accurate aging of the stage of pregnancy but is not capable of reliable detection of multiple pregnancies. External palpation of the uterus and uterine environment (through the body wall of the mother) may allow detection of pregnancy; but at a later stage of pregnancy and at lower sensitivity than is possible using internal palpation based methods. The operator skill required to undertake accurate palpation-based pregnancy diagnosis is extensive and the process is often limited by law to veterinarians or medical practitioners within many jurisdictions.
Another physical method for pregnancy detection is the use of ultrasound (US) to detect the presence of a foetus and/or changes to the uterine environment associated with pregnancy (including size, fluid content and blood flow). Ultrasound-based methods transmit high frequency sound waves towards structures under examination and record reflected sound waves. These reflected waves are processed and usually converted to visual images for assessment by the operator.
One of the US methods employed is two-dimensional real time US which uses a transducer array to create an image of the structures within the projected (and reflected) sound beam. This method requires skill to direct the transducer to the appropriate site, to exclude interposed air pockets and to interpret signal and images that are obtained.
Another US method is RT-Mode US which allows identification of foetal age, the presence of foetal death, multiple foetuses (i.e. litters) and sexing of foetuses. High frequency transducers are needed to visualise delicate uterine and foetal structures. These higher frequencies are characterised by poor tissue penetration, necessitating close internal positioning of the probe and highly skilled operators.
A (Amplitude)-Mode US uses a reflected sound wave to determine the depth of tissue structures below the probe. Again, significant skill is required to move the device into the appropriate position and to interpret the output.
B (Brightness)-Mode US is similar to A mode ultrasound except the reflected sound wave is converted to light of an intensity that mimics the amplitude of the reflected sound. A- and B-Mode US methods have been described in pregnancy diagnosis in animals.
M Mode US uses a single beam of sound and the reflected signal is used to update a visual presentation of movement of the interfaces between surfaces continually. The frequency of transmitted sound waves is typically very high and therefore this method is useful to visualise rhythmic movements (e.g. the heart) and for assessing size of structures. M mode is used extensively in human cardiac and foetal cardiac imaging.
Doppler US measures the change in frequency that occurs when transmitted sound waves are reflected from moving surfaces (or fluids). This change in frequency is converted to an audible signal that the operator may use to identify movement (such as due to the presence of a foetus). The foetal heart and circulatory movement can be detected with this method along with changes to maternal uterine blood flow as blood flow increases to meet foetal and placental demand. Doppler US probes must be directed against the structure in question and at an angle that is not perpendicular to the plane of movement. As such, reasonable skill is required to produce a diagnostic signal. Doppler US allows identification of multiple foetuses (e.g. the detection of multiparous pregnancies and estimated litter size). Doppler US has not proved reliable in pigs or cattle pregnancy diagnosis being associated with low sensitivity.
Another potential physical detection method for pregnancy diagnosis is the electrocardiogram (ECG). An ECG has been used to record foetal heart activity with detection of the bovine foetal heart from 150 days of pregnancy reported. Detection time can be slow (one study reporting between 3-5 minutes to obtain a recording from pregnant cows in the second half of gestation) as the raw signal must be visually examined for presence of foetal heart activity. Sensitivity of the method is not optimal but the method can reliably detect multiple pregnancies in cattle and horses.
The foetal ECG signal from external leads applied to the mother in late pregnancy in cattle is around 10 μV and significant electrical noise from the farm environment (e.g. from milking machines, electrical fences, etc.) is often present. A study in horses found the variability in signal strength arising from different lead positions and the numerous lead configurations to be the greatest challenge to the use of ECG. The study also found great variability in foetal heart rate in horses; in contrast to cattle. Other studies have found equine foetal heart rate monitoring using ECG successful but time consuming to obtain. Twin foetuses are readily detected using external ECG in horses. Electrode attachment to the skin of the mother is not without risk. The abdominal electrode especially is resented by many mares necessitating movement to a more lateral position, away from the ventral midline.
The maternal ECG of dairy cows may also experience changes associated with pregnancy. A standard six-limb ECG identified two significant findings: (1) for lead II, the T-wave is negative for most pregnant cows and positive for non-pregnant cows and (2) pregnant cows generally demonstrate a right axis deviation in the mean electric axis when compared to non-pregnant cows (i.e. no axis deviation).
Laboratory-based analytical methods of pregnancy diagnosis require the collection of a diagnostic sample obtained from the mother (e.g. blood, urine, milk, faeces). The sample is analysed to determine the presence of or level of a specific hormone or chemical (metabolite) that is correlated to pregnancy status and, for some, to the stage of the pregnancy. These methods require significant efficiencies in sample collection, identification, processing and reporting to minimise time lag and identification errors. Many are intrusive, requiring invasive sample collection (e.g. blood). Many assays require laboratory support and/or prolonged sample processing time and are therefore not able to provide a diagnosis to the operator within a few minutes of collection of the sample. This lack of timeliness prevents immediate processing of animals based upon pregnancy result and necessitates at least two collection, handling and processing events for the livestock. Many assays are expensive and therefore are not practical for commercial farm animals. Sample-based systems are also prone to transcription errors—especially for tests requiring more than one livestock processing event. There are numerous laboratory sample-based methods for diagnosing pregnancy. None has the necessary accuracy, convenience, timeliness of diagnosis and cost effectiveness for commercial use on animals. All require either use of laboratories or prolonged field processing times meaning that results are not available in real time. Many assays measure compounds that persist for prolonged periods (up to 100 days) after birth making the assessment of subsequent pregnancies difficult and thereby reducing specificity.
The disadvantages of existing methods of pregnancy diagnosis are many. Manual-based methods require significant operator skill and often make use of expensive equipment. As such, these diagnostic methods are typically provided by contractors on a fee-for-service basis. Often the supply of contractors is limited within regions (and in many jurisdictions supply is limited by acts of law to medical practitioners and veterinarians). Manual-based methods may also present a risk to the unborn foetus, the animal and the operator. Laboratory-based methods also require sample collection and this can be invasive, inconvenient and expose the animal to risk (infection, haemorrhage). Most assay-based methods do not provide a diagnosis in real time and are costly.
To the best of the Applicants' knowledge, all existing forms of pregnancy diagnosis either require access to expertise and/or specialised equipment thereby limiting their convenience. Many require a time delay (assay-based testing) and all are associated with ongoing costs. Given the current constraints, most pregnancy diagnosis of farms animals is therefore performed on batches of animals when the cost of intervention can be spread across more than one individual. Very rarely are single animal diagnoses sought resulting in delayed testing of individuals.